Comparison of Phytochemical and Antioxidant Activities in Micropropagated and Seed-derived Salvia miltiorrhiza Plants

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  • 1 College of Life Sciences, Qufu Normal University, Qufu, Shandong 273165, China

Salvia miltiorrhiza (commonly known in China as Danshen) is widely used in traditional Chinese medicine, and it is applied in the treatment of many diseases, particularly cardiovascular disease. Commercial propagation of Danshen is carried out either through seed germination or in vitro regeneration (micropropagation). However, it is not clear if the different propagation methods affect the chemical properties of the derived plants. In the present study, we first established a highly efficient tissue culture system for Danshen propagation. The addition of 1.0 mg·L−1 6-benzyladenine (BA) and 0.1 mg·L−1 α-naphthalene acetic acid (NAA) to Murashige and Skoog (MS) medium was optimal for inducing adventitious shoots; the highest rate of rooting was recorded on MS medium with 0.2 mg·L−1 NAA, on which the survival rate of transplanted plantlets was 95%. Next, we assessed antioxidant properties in the different tissues of plants of the same age, derived from micropropagation or seed germination, and measured tanshinone, total phenol, and total flavonoid contents. Our results showed that tissues of micropropagated plantlets had higher antioxidant activities than tissues of seed-derived plantlets; the micropropagated plantlets also had higher tanshinone contents in their roots. Thus, a rapid and efficient micropropagation system was established for Danshen, and it can be used for cultivating this plant to obtain therapeutic compounds.

Abstract

Salvia miltiorrhiza (commonly known in China as Danshen) is widely used in traditional Chinese medicine, and it is applied in the treatment of many diseases, particularly cardiovascular disease. Commercial propagation of Danshen is carried out either through seed germination or in vitro regeneration (micropropagation). However, it is not clear if the different propagation methods affect the chemical properties of the derived plants. In the present study, we first established a highly efficient tissue culture system for Danshen propagation. The addition of 1.0 mg·L−1 6-benzyladenine (BA) and 0.1 mg·L−1 α-naphthalene acetic acid (NAA) to Murashige and Skoog (MS) medium was optimal for inducing adventitious shoots; the highest rate of rooting was recorded on MS medium with 0.2 mg·L−1 NAA, on which the survival rate of transplanted plantlets was 95%. Next, we assessed antioxidant properties in the different tissues of plants of the same age, derived from micropropagation or seed germination, and measured tanshinone, total phenol, and total flavonoid contents. Our results showed that tissues of micropropagated plantlets had higher antioxidant activities than tissues of seed-derived plantlets; the micropropagated plantlets also had higher tanshinone contents in their roots. Thus, a rapid and efficient micropropagation system was established for Danshen, and it can be used for cultivating this plant to obtain therapeutic compounds.

The genus Salvia (family Lamiaceae) consists of nearly 1000 species (Claßen-Bockhoff et al., 2004); many of them are used as herbs or traditional medicines because they contain active compounds that function against tumors, inflammation, and cardiovascular disease (Li et al., 2015; Wang et al., 2013). Species within the genus Salvia are also exploited for food, spices, cosmetics, and ornamental purposes (Neugebauerová et al., 2015; Wang et al., 2011). The dried roots of S. miltiorrhiza, known as Danshen in China, contains the pharmaceutically important secondary metabolites tanshinone and methyltanshinone, and are widely used in coronary heart disease therapeutics, especially for treating angina pectoris and myocardial infarction in China, Japan, Korea, and other Asian countries (Ryu et al., 1996; Wang et al., 2011). Because of its medicinal value and increasing demand, wild resources of S. miltiorrhiza have been subjected to excessive exploitation and the species has become threatened; for this reason, cultivated Danshen is generally used for clinical purposes. However, the yield and quality of active compounds from Danshen decrease after prolonged field cultivation; in addition, viral infections and uncontrolled cross-pollination in the field may lead to varietal loss (Shan et al., 2007). In recent years, methodological advances in plant tissue culture methods have allowed its use as a viable approach for multiplication, germplasm conservation, and genetic manipulation of medicinal plants (Benson, 2008; Gonçalves et al., 2010). Hence, tissue culture may offer an efficient alternative method to improve the quality and yield of active compounds in Danshen.

The importance of Danshen in traditional medicine has stimulated the development of tissue culture methods for the production of tanshinone, such as callus and cell cultures, and the stimulation of adventitious and hairy roots (Chen et al., 2001; Hu and Alfermann, 1993; Shan et al., 2007; Zhao et al., 1999). Although tanshinone production from S. miltiorrhiza has improved (Shi et al., 2007; Wu and Shi, 2008; Zhang et al., 2004; Zhao et al., 2010), to the best of our knowledge, the relative contents of tanshinone in extracts of shoots and roots grown in vitro have not been compared with that of extracts from plantlets of the same age but derived from seeds. The present study was initiated to answer this question and to examine the antioxidant activities of tissues from in vitro–grown plants and determine the free radical scavenging properties of S. miltiorrhiza extracts. Antioxidants can alleviate oxidative damage induced by reactive oxygen species (ROS) (Zhu et al., 2004). In general, most of the important medicinal bioactive secondary metabolites are phenols and flavonoids that inhibit life-threatening degenerative diseases, e.g., cardiovascular disease, cancer, and neurological disorders, which are induced by oxidative stress (Kinsella et al., 1993; Reddy et al., 2012). In plants, high level of oxidative stress results in an increased production of ROS, which have a harmful effect on plant cells. The most important forms of ROS are hydrogen peroxide (H2O2), superoxide (O2), and nitric oxide (NO) (Kumaran and Karunakaran, 2007). Increases in the levels of these ROS can cause plant cell death through oxidation stress and denaturation of macromolecules, such as proteins, DNA, and unsaturated fatty acids (Halliwell and Gutteridge, 2007). Therefore, identifying which plant tissues contain the highest contents of antioxidant compounds might lead to the identification and characterization of novel compounds for medical applications.

In this study, we explored the effects of various cytokinins and an auxin on the induction of adventitious shoots in S. miltiorrhiza. Proliferating shoots were then rooted and acclimatized in a greenhouse. The antioxidant characteristics, phenol and flavonoid concentrations, and tanshinone content of in vitro–regenerated (micropropagated) plants in different tissues were compared with that from plants of the same age propagated from seeds.

Materials and Methods

Plant materials and culture conditions.

Salvia miltiorrhiza plants were obtained from Shilai town, Taian city in Shandong Province, China, and cultivated in a greenhouse belonging to the Qufu Normal University, Shandong Province, China. Healthy leaves, which were selected as explants, were disinfected using 75% (v/v) ethanol for 30 s and then rinsed three times with sterile distilled water. Thereafter, the explants were sterilized using 0.1% (w/v) aqueous HgCl2 for 7 min followed by four rinses with sterile distilled water. The base medium used here was MS medium with 3% (w/v) sucrose and 0.65% (w/v) agar. Medium pH was adjusted to 5.9 using 0.1 n NaOH or 0.1 n HCl, and the medium was autoclaved at 121 °C for 20 min. All cultures were placed at 25 ± 1 °C under a 12-h photoperiod with 40 μmol·m−2·s−1 photosynthetic photon flux density from cool-white fluorescent lamps (Philips 40 W tubes, NVC lighting, Huizhou, China).

Adventitious shoot induction.

Sterilized leaf explants were inoculated on MS medium containing various concentrations (0, 0.5, 1.0, 2.0, or 3.0 mg·L−1) of BA, kinetin (KT), or thidiazuron (TDZ). Thereafter, BA was chosen as the optimum cytokinin for evaluation and tested in a mixture with the auxin NAA at several concentrations (0.1, 0.2, 0.3, or 0.4 mg·L−1) for its ability to induce adventitious shoot formation. While TDZ was filter-sterilized and then added to the sterilized medium, BA, KT, and NAA were added to the MS medium before pH adjustment and the sterilization step. After 6 weeks of culture, the rate of shoot induction, the number of shoots induced per plant, and the lengths of shoots were determined. Thirty explants were employed and each treatment was repeated three times.

Rooting and acclimatization.

After adventitious shoot induction, elongated shoots (2–4 cm) were inoculated on MS medium containing different concentrations of NAA (0, 0.1, 0.2, 0.3, or 0.4 mg·L−1) to induce root formation. After 3 weeks, the rate of root formation, and the length and number of roots were assessed. Plantlets with roots were transferred to 8-cm-diameter pots containing a mixture of vermiculite and soil (1:1, v/v; flower nutrition soil; Deli, Fuzhou, China) and grown in a greenhouse with day/night temperatures of 25/18 °C and 75% to 80% relative humidity. The survival rate of the transplants was calculated at 4 weeks of acclimatization.

Sample preparation, phytochemical compounds extraction, and analysis of total phytochemical compounds.

For phytochemical evaluation, shoot and root samples (0.5 g) were collected from plants derived from adventitious small shoots cultured on optimal rooting media after 10 weeks and from seed-derived S. miltiorrhiza plants that had been grown for 10 weeks in soil. The samples were ground in liquid nitrogen and then extracted with 5 mL 80% (v/v) methanol with shaking at 500 rpm for 4 h (García-Pérez et al., 2012). Thereafter, the extracts were centrifuged at 7000 rpm (D-37520; Heraeus-Thermo, Osterode, Germany) for 10 min and the supernatant was employed in phytochemical analysis.

The total phenol content of the extracts was evaluated following the theory of Folin–Ciocalteu (Singleton et al., 1999). Partial extracts (25 μL) were diluted to 2 mL with distilled water and then added to Folin–Ciocalteu reagent (0.5 mL). The mixture was placed in the dark for 5 min. Then, 1 mL 5% (w/v) Na2CO3 was added, the mixture was diluted to 5 mL with distilled water, and then incubated at 25 °C for 1 h. The reaction absorbance was recorded at 750 nm using an ultraviolet-5500 spectrophotometer (Shanghai Metash Instruments, Shanghai, China) and the content of total phenol was expressed as the content of gallic acid equivalents.

Total flavonoid content was evaluated using the aluminum chloride calorimetric method with slight modification (Kim et al., 2003). Samples (1 mL) were diluted to 4 mL with methanol (80%) and added to 0.3 mL 5% (w/v) NaNO2 solution. The reaction mixture was kept at room temperature (25 °C) for 5 min. After this period, 0.3 mL 10% (w/v) AlCl3 solution was added; after 6 min, 4 mL NaOH (4%, w/v) was added to the mixture. Finally, the reaction mixture was diluted to 10 mL with distilled water. The absorbance of the reaction mixture was recorded at 510 nm using an ultraviolet-5500 spectrophotometer and the total flavonoid content of each extract was calculated from a standard rutin calibration curve.

Quantification of total tanshinone using the methanol refluxing method.

Plant samples (6.0 g) were lyophilized and macerated and then extracted twice using the methanol (100%, 60 mL) reflux extraction method; each extraction was performed for 2 h (Xu and Xiang, 2014). The extracted liquid was added to a 250-mL volumetric flask, brought to volume (250 mL) with methanol (100%), and shaken until fully mixed. A 5-mL aliquot of this mixture was then placed in a volumetric flask (50 mL) and brought to 50 mL with methanol (100%). After attenuation, absorbance was recorded at 269 nm using an ultraviolet-5500 spectrophotometer, and the total tanshinone content was calculated from a standard tanshinone IIA calibration curve (Lan et al., 2012; Li et al., 2012).

Antioxidant ability of plant extracts.

Shoots and roots of micropropagated and seed-derived plants were ground in a solution containing phosphate buffer solution (PBS, 50 mm), 20% (v/v) glycerin, DL-dithiothreitol (1 mm), ethylenediaminetetraacetic acid (EDTA-Na2, 1 mm), and MgSO4 (1 mm). Briefly, the plant samples (0.5 g) were homogenized to a powder and then extracted with 5 mL of the solution described previously. The resulting suspension was centrifuged at 7000 rpm (10 min) and then at 10,000 rpm (15 min). The supernatant was used for the in vitro trials described next.

Scavenging activity of O2 radicals.

The scavenging activity assay was based on the ability of extracts to inhibit formazan production by bleaching the O2 radicals generated in nitro blue tetrazolium (NBT) salt with riboflavin and light (Kumaran and Karunakaran, 2007). The extracts (0.1 mL) were added to a reaction mixture (0.1 mg NBT, 100 μM EDTA-Na2, and 50 mm sodium phosphate buffer containing 20 μg riboflavin; pH 7.6) and illuminated. After 20 min, the absorbance was recorded at 560 nm using an ultraviolet-5500 spectrophotometer.

Scavenging activity of H2O2 radicals.

For the H2O2 scavenging experiment, extracts (0.1 mL) were added to the reaction mixture [1.2 mL distilled water and 0.2 mL H2O2 (100 mm) in 1.5 mL sodium phosphate buffer; pH 7.0]. After 3 min, the absorbance was recorded at 240 nm using an ultraviolet-5500 spectrophotometer against a blank solution that lacked H2O2 (Kumaran and Karunakaran, 2007).

Scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals.

For the DPPH scavenging experiment, sample extracts (40 μL) were first mixed with 1960 μL methanolic solution (0.1 mm) of DPPH; this mixture was incubated for 25 min in the dark before determining its absorbance at 517 nm using an ultraviolet-5500 spectrophotometer (Manivannan et al., 2015).

Scavenging activity of NO radicals.

Inhibition of NO generation by plant extracts was estimated using sodium nitroprusside (SNP)–mediated NO production (Manivannan et al., 2015). Nitric oxide is produced spontaneously when SNP reacts with oxygen to produce nitrite ions, which can be estimated using Griess reagent. The reaction started when SNP (10 mm) in PBS was added to 0.1 mL extracts. The mixture was placed at 25 °C for 150 min and 0.5 mL freshly prepared Griess reagent [1% sulfanilic acid, 2% H3PO4, and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride] was added to the mixture after this period. Absorbance was determined at 546 nm using an ultraviolet-5500 spectrophotometer.

Free radical scavenging was assayed using ascorbic acid (AA). The rate of radical scavenging (%) was measured using the formula [(AcAs)/Ac] × 100, where Ac is the optical density (OD) value of the control (which lacked the extracts), and As is the OD of the extracts or AA.

Statistics.

All assays were performed using a stochastic experimental design with three replication treatments, and each experiment was repeated three times to confirm the repeatability of results. The significance of differences between treatments was tested by analysis of variance followed by Duncan’s multiple range tests at a 5% significant level, using the SPSS software package (IBM, Armonk, NY).

Results and Discussion

Effect of plant growth regulators (PGRs) on shoot induction.

Plant cytokinins and auxins are important regulators in plant differentiation (Frello et al., 2002; Kordi et al., 2013; Peeters et al., 1991). In the present study, we examined the abilities of various PGRs and their concentrations to induce adventitious shoots from leaf explants of field-growing plants. Healthy leaf explants were cultured on MS medium with or without PGRs to induce shoot propagation. Explants on MS medium without PGRs produced only a few shoots. However, explants on MS medium containing a cytokinin formed several adventitious shoots (Table 1). Explants first showed differentiation at 12–14 d of culture on cytokine-supplemented medium and eventually formed green callus (Fig. 1A). After 4 weeks, the regeneration of shoots was recognized by the formation of small leaf-like tissues on the leaf edge that subsequently produced adventitious shoots (Fig. 1B).

Table 1.

Effect of different concentrations of plant growth regulators on adventitious shoot induction from the leaf explants of Salvia miltiorrhiza.

Table 1.
Fig. 1.
Fig. 1.

Micropropagation of Salvia miltiorrhiza. (A) Green-colored protuberances from leaf explants; (B) Adventitious shoots induced after 3 weeks; (C) Shoot proliferation and elongation; (DG) The plantlets rooted from the shoot base; (H) Acclimatized plants in the greenhouse (bars = 1 cm).

Citation: HortScience horts 53, 7; 10.21273/HORTSCI13072-18

The highest rate (83.3%) of shoot induction and largest number of shoots (10.3) were achieved on MS medium containing 1.0 mg·L−1 BA (Table 1). This finding is consistent with previous reports on BA being effective for in vitro proliferation and multiplication of S. miltiorrhiza (Xie et al., 2004; Zhao et al., 1999), Salvia fruticosa (Arikat et al., 2004), and Salvia leucantha (Hosoki and Tahara, 1993). However, an increase in concentration to 2.0 mg·L−1 BA resulted in a decrease in the rate of shoot induction and formation of brown calli on leaf edges. This result could be due to BA metabolism and induction of other endogenous hormones in the plant tissues (Sharma and Wakhlu, 2003). Most adventitious shoots on media with different concentrations of TDZ showed hyperhydricity, resulting in their malformation and translucent or glassy appearance, with low lignification and poor regeneration. Hyperhydricity typically occurs under stress conditions during tissue culture but has been reported after application of high concentrations of PGRs such as TDZ (Caboni et al., 1999; Ghimire et al., 2012). Overall, the combination of cytokinin analogs and auxin improved the rate of adventitious shoot induction (Burdyn et al., 2006; Ghimire et al., 2012; Zheng et al., 2009). In the present study, MS media containing BA (1.0 mg·L−1) and NAA (0.1, 0.2, 0.3, or 0.4 mg·L−1) showed a concentration-related increase in the rate of shoot regeneration compared with BA alone (Table 2). The largest number of shoots per explant (22.3) was found on MS medium supplemented with 1.0 mg·L−1 BA and 0.1 mg·L−1 NAA (Fig. 1C), whereas the highest rate of shoot induction (100%) was observed on MS medium supplemented with 1.0 mg·L−1 BA and 0.2 mg·L−1 NAA [although fewer shoots per explants (11.0) were seen on this medium].

Table 2.

Effect of 6-benzyladenine (BA) and α-naphthalene acetic acid (NAA) combination on adventitious shoot induction of Salvia miltiorrhiza.

Table 2.

Effect of NAA on rooting and acclimatization.

Plant auxins such as indole-3-butyric acid, indole acetic acid, or NAA have been applied to induce root formation in tissue explant cultures (Caboni et al., 1999). In our previous study, the addition of 0.05 mg·L−1 NAA to MS medium resulted in efficient rooting in Haworthia turgida explants (Liu et al., 2017). Here, healthy shoots (2–4 cm in length) were cut from elongated shoots and cultured on MS media with NAA (0, 0.1, 0.2, 0.3, or 0.4 mg·L−1) to induce rooting. Roots emerged from the shoot base within 10 d of culture on rooting medium and efficient induction of the root system was observed at three weeks of culture (Fig. 1D and E). The highest rate of rooting was achieved with 0.2 mg·L−1 NAA, as 96.7% of the explanted shoots showed induced root formation, with an average root length of 34.7 mm and an average root number of 5.3 (Table 3). Well-rooted plantlets (Fig. 1F and G) were moved to pots containing nutritive soil, placed within the greenhouse, and generated true leaves after ≈10 d. In general, a 95% survival rate was achieved at 4 weeks within the greenhouse (Fig. 1H).

Table 3.

Effect of α-naphthalene acetic acid (NAA) on root induction of Salvia miltiorrhiza.

Table 3.

Assessment of phytochemicals

Determination of total phenol and flavonoid content.

Natural phenolic and flavonoid compounds are known to have positive effects on inflammation and cardiovascular disease and to show antioxidant activities (Alothman et al., 2009; Jagtap et al., 2011; Xanthopoulou et al., 2010). The properties of the different phytochemical components of different tissues of S. miltiorrhiza are essential to its medicinal properties. Here, we determined the total phenol and flavonoid contents of extracts from shoots and roots of regenerated plants, and shoots and roots of seed-derived plants (Fig. 2). Overall, shoot extracts had higher total flavonoid (Fig. 2A) and phenol contents (Fig. 2B) than root extracts, regardless of whether they were from plants derived from in vitro culture or from seeds. Similar results have been reported for Aloe arborescens and Psoralea drupacea (Amoo et al., 2012; Lystvan et al., 2010). The aboveground parts of plants are often adapted to a higher rate of synthesis of secondary metabolites than underground tissues (Bourgaud et al., 2001). The roots of micropropagated plants had higher phenol contents than those of seed-derived plants. The total flavonoid contents were upregulated in the shoots of plant micropropagated in a medium with PGRs, but did not differ significantly in the roots of the two plant groups (Fig. 2A). The variations in flavonoid and phenolic contents in different tissues could be due to disparities in tissue formation and accumulation of phytochemicals, plant physiology, or inherent hormone levels (Surveswaran et al., 2010). Previous studies reported that different types and contents of PGRs have modified the contents of secondary metabolites in plants (Baskaran et al., 2012; Palacio et al., 2008). Baskaran et al. (2014) reported that PGRs had a significant effect on the synthesis and accumulation of phenolic compounds and flavonoids in in vitro–derived plants of Coleonema pulchellum. Nevertheless, our results demonstrate that the contents of phytochemicals in micropropagated and seed-derived plants were similar, and thus that micropropagated plants might be used as a substitute of seed-derived plants when exploited for their medicinal benefits.

Fig. 2.
Fig. 2.

Phytochemical contents of extracts from micropropagated and seed-derived Salvia miltiorrhiza plants. Total flavonoid (A) and total phenol (B) contents present in shoot (IS) and root (IR) extracts of micropropagated plants, and shoot (SS) and root (SR) extracts of seed-derived plants. Different capital letters among bars indicate statistically significant differences at P ≤ 0.05, according to Duncan’s multiple range test.

Citation: HortScience horts 53, 7; 10.21273/HORTSCI13072-18

Assessment of total tanshinone content in tissue extracts.

The level of tanshinone was identified in tissue extracts (Fig. 3). The highest level of tanshinone was 8.77 μg·mg−1 fresh weight (FW) in roots of micropropagated plants and 8.52 μg·mg−1 FW in roots of seed-derived plants. The lowest level of tanshinone was 3.18 μg·mg−1 FW in shoot extracts of seed-derived plants and 3.46 μg·mg−1 FW in shoots extracts of micropropagated plants. The slight increase in tanshinone in micropropagated plants might be due to the plant tissue culture environment or to the effect of endogenous hormones. Previous studies reported that the in vitro environment might increase the production of secondary metabolites by modifying primary metabolism (Close and McArthur, 2002; Gould et al., 2000). In addition, levels of secondary metabolites may be greatly affected by the nutrients and plant hormones used during tissue culture (Baskaran et al., 2012). Higher production of secondary metabolites in micropropagated plants than in natural populations has also been reported in Swertia japonica (Ishimary et al., 1990) and Gentiana lutea (Menković et al., 2000).

Fig. 3.
Fig. 3.

Tanshinone content of extracts from micropropagated and seed-derived Salvia miltiorrhiza plants. The tanshinone content of shoot (IS) and root (IR) extracts of micropropagated plants, and shoot (SS) and root (SR) extracts of seed-derived plants. Different capital letters among bars indicate statistically significant differences at P ≤ 0.05, according to Duncan’s multiple range test.

Citation: HortScience horts 53, 7; 10.21273/HORTSCI13072-18

Free radical scavenging ability

Scavenging ability of O2 radical.

Extracts from all tissues inhibited the production of blue formazan by scavenging O2 radicals produced by the NBT–riboflavin–light complex. The relative scavenging ability of each tissue extract was compared with that of a control and AA (Fig. 4A). Although all extracts efficiently inhibited O2, roots of micropropagated plants showed the highest scavenging activity (77.48%). Furthermore, significant differences were found for the relative O2 scavenging abilities of shoots between micropropagated plants (71.47%) and seed-derived plants (65.22%). The rate of AA scavenging was 77.23%. Superoxide radicals are greatly detrimental to plant cell organelles and can act as precursors for ROS production (Dewir et al., 2006; Manivannan et al., 2015). In addition, excess production of O2 in cells can enhance dismutation, which results in the production of H2O2 that further increases oxidative stress (Halliwell and Gutteridge, 2007). Therefore, plant cells require an efficient antioxidant system to scavenge O2.

Fig. 4.
Fig. 4.

Free radical scavenging ability of extracts from micropropagated and seed-derived Salvia miltiorrhiza plants. Superoxide (A), hydrogen peroxide (B), 2,2-diphenyl-1-picrylhydrazyl radical (C), and nitric oxide (D) scavenging activities of shoot (IS) and root (IR) extracts of micropropagated plants, and shoot (SS) and root (SR) extracts of seed-derived plants and of ascorbic acid (AA). Different capital letters among bars indicate statistically significant differences at P ≤ 0.05, according to Duncan’s multiple range test.

Citation: HortScience horts 53, 7; 10.21273/HORTSCI13072-18

Scavenging ability of H2O2 radicals.

The capacity of tissue extracts to suppress H2O2 radicals was compared with that of AA (Fig. 4B). The rates of H2O2 scavenging ranged from 48.56% to 85.45%. As expected, AA was the most efficient scavenger (85.46%), followed by shoots of micropropagated plants (85.45%) and shoots of seed-derived plants (80.61%). Root extracts showed moderate scavenging activity: 55.59% in micropropagated plants and 48.56% in seed-derived plants. In general, H2O2 is nonreactive, but it can cause the generation of other harmful free radicals, such as hydroxyl radicals, and result in plant cell death. Therefore, it is essential for cells to possess an efficient antioxidant system to scavenge H2O2 radicals and optimize the cellular environment. Previous reports indicated that phenolic compounds can act as scavengers of H2O2 (Halliwell and Gutteridge, 2007). Therefore, the existence of a high level of phenolic compounds might have contributed to the improved H2O2 scavenging capacity of micropropagated plants.

Scavenging ability of DPPH radicals.

The DPPH assay has been generally used to determine the antioxidant capacity of extracts from in vitro–regenerated plant tissues (Alothman et al., 2009). Here, all extracts exhibited DPPH radical scavenging ability (Fig. 4C). The highest rate of DPPH scavenging was found in shoots of micropropagated plants (84.68%), with a lower rate in shoots of seed-derived plants (75.38%). In root extracts, scavenging rates of 63.78% and 62.50% were found for micropropagated and seed-derived plants, respectively. Because DPPH is a purple-colored molecule that turns yellow after receiving an electron from an antioxidant and can be spectrophotometrically recorded, the level of antioxidants in a mixture containing DPPH can be assessed by its level of discoloration (Surveswaran et al., 2010). Ahmad et al. (2010) reported increased radical scavenging activity in micropropagated shoots of Piper nigrum.

Scavenging ability of NO radicals.

All extracts tested here showed ability to scavenge NO radicals (Fig. 4D). Extracts from shoots of micropropagated and seed-derived plants showed a similar rate of NO radicals scavenging: 83.48% and 83.54%, respectively. Moreover, NO inhibition in roots from micropropagated and seed-derived plants were 65.54% and 65.30%, respectively (Fig. 4D). In biological systems, excess production of NO radicals is associated with a range of diseases, such as cancer, chronic inflammatory disorders, and atherosclerosis (Moncada et al., 1991). Cells, therefore, require the ability to reduce excess production of NO radicals to prevent disease.

Conclusions

The present study developed an efficient propagation system for S. miltiorrhiza. The highest efficiency of shoot induction was obtained on MS medium with 1.0 mg·L−1 BA and 0.1 mg·L−1 NAA, and MS medium supplemented with 0.2 mg·L−1 NAA was the best for S. miltiorrhiza rooting. This study also demonstrated that the tissues of micropropagated S. miltiorrhiza contain abundant flavonoid and phenolic compounds and that extracts showed scavenging activities to commonly formed cellular free radicals. The information provided here will be of importance to large-scale micropropagation of S. miltiorrhiza for commercial purposes and for germplasm conservation. Moreover, the tanshinone contents and scavenging potential of free radicals in S. miltiorrhiza tissue extracts reported here will be of value for therapeutic applications. The difficulty of maintaining high-quality plants from seeds could be circumvented by using plant tissue culture, without affecting the medicinal value of S. miltiorrhiza.

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    • Export Citation
  • García-Pérez, E., Janet, A., Uribe, G. & García-Lara, S. 2012 Luteolin content and antioxidant activity in micropropagated plants of Poliomintha glabrescens (Gray) Plant Cell Tissue Organ Cult. 108 521 527

    • Search Google Scholar
    • Export Citation
  • Ghimire, B.K., Yu, C.Y. & Chung, I.M. 2012 Direct shoot organogenesis and assessment of genetic stability in regenerants of Solanum aculeatissimum Jacq Plant Cell Tissue Organ Cult. 108 455 464

    • Search Google Scholar
    • Export Citation
  • Gonçalves, S., Fernandes, L. & Romano, A. 2010 High-frequency in vitro propagation of the endangered species Tuberaria major Plant Cell Tissue Organ Cult. 101 359 363

    • Search Google Scholar
    • Export Citation
  • Gould, K.S., Markham, K.R., Smith, R.H. & Goris, J.J. 2000 Functional role of anthocyanins in the leaves of Quintinia serrata A Cunn. J. Expt. Bot. 51 1107 1115

    • Search Google Scholar
    • Export Citation
  • Halliwell, B. & Gutteridge, J.M.C. 2007 Free radicals in biology and medicine, Vol. 3. 4th ed. Oxford Univ. Press, Oxford, UK

  • Hosoki, T. & Tahara, Y. 1993 In vitro propagation of Salvia leucantha Cav HortScience 28 226

  • Hu, Z.B. & Alfermann, A.W. 1993 Diterpenoid production in hairy root cultures of Salvia miltiorrhiza Phytochemistry 32 699 703

  • Ishimary, K., Sudo, H., Satake, M., Matsunaga, Y., Hasegawa, Y., Takemoto, S. & Shimomura, K. 1990 Amarogentin, amaroswerin and four xanthones from hairy root cultures of Swertia japonica Phytochemistry 29 1563 1565

    • Search Google Scholar
    • Export Citation
  • Jagtap, U.B., Waghmare, S.R., Lokhande, V.H., Suprasanna, P. & Bapat, V.A. 2011 Preparation and evaluation of antioxidant capacity of jackfruit (Artocarpus heterophyllus Lam.) wine and its protective role against radiation induced DNA damage Ind. Crops Prod. 34 1595 1601

    • Search Google Scholar
    • Export Citation
  • Kim, D.O., Chun, O.K., Kim, Y.J., Moon, H.Y. & Lee, C.Y. 2003 Quantification of polyphenolics and their antioxidant capacity in fresh plums J. Agr. Food Chem. 51 6509 6515

    • Search Google Scholar
    • Export Citation
  • Kinsella, J.E., Frankel, E., German, B. & Kanner, J. 1993 Possible mechanisms for the protective role of antioxidants in wine and plant foods Food Technol. 47 85 89

    • Search Google Scholar
    • Export Citation
  • Kordi, M., Kaviani, B. & Hashemabadi, D. 2013 In vitro propagation of Kalanchoe blossfeldiana using BA and NAA Eur. J. Expt. Biol. 3 285 288

  • Kumaran, A. & Karunakaran, R.J. 2007 In vitro antioxidant activities of methanol extracts of five Phyllanthus species from India LWT-Food Sci. Technol. 40 344 352

    • Search Google Scholar
    • Export Citation
  • Lan, T.F., Wang, X., Wang, D.J., Wang, L., Liu, Q. & Yu, Z.Y. 2012 Determination of four tanshinones in Salvia miltiorrhiza Radix et Rhizoma with quantitative analysis of multi-components by single-marker Chin. Tradit. Herbal Drugs 43 2420 2423 (in Chinese)

    • Search Google Scholar
    • Export Citation
  • Li, B., Zhang, C.L., Peng, L., Liang, Z.S., Yan, X.J., Zhu, Y.H. & Liu, Y. 2015 Comparison of essential oil composition and phenolic acid content of selected Salvia species measured by GC-MS and HPLC methods Ind. Crops Prod. 69 329 334

    • Search Google Scholar
    • Export Citation
  • Li, Q., Liu, W., Luo, Z.L. & Yang, M.H. 2012 Simultaneous determination of four tanshinones in Salvia miltiorrhiza by QAMS Chian J. Chinese Mater. Med. 37 824 828 (in Chinese)

    • Search Google Scholar
    • Export Citation
  • Liu, B.L., Fang, H.Z., Meng, C.R., Chen, M., Chai, Q.D., Zhang, K. & Liu, S.J. 2017 Establishment of a rapid and efficient micropropagation system for succulent plant Haworthia turgida Haw HortScience 52 1 5

    • Search Google Scholar
    • Export Citation
  • Lystvan, K., Belokurova, V., Sheludko, Y., Ingham, J.L., Prykhodko, V., Kishchenko, O., Paton, E. & Kuchuk, M. 2010 Production of bakuchiol by in vitro systems of Psoralea drupacea Bge Plant Cell Tissue Organ Cult. 101 99 103

    • Search Google Scholar
    • Export Citation
  • Manivannan, A., Soundararajan, P., Halimah, N., Chung, H.K. & Jeong, B.R. 2015 Blue LED light enhances growth, phytochemical contents, and antioxidant enzyme activities of Rehmannia glutinosa cultured in vitro Hort. Environ. Biotechnol. 56 105 113

    • Search Google Scholar
    • Export Citation
  • Menković, N., Fodulović, S.K., Momcilović, I. & Grubisić, D. 2000 Quantitative determination of secoiridoid and gamma-pyrone compounds in Gentiana lutea cultured in vitro Planta Med. 66 96 98

    • Search Google Scholar
    • Export Citation
  • Moncada, S., Palmer, R.M. & Higgs, E.A. 1991 Nitric oxide: Physiology, pathophysiology, and pharmacology Pharmacol. Rev. 43 109 142

  • Neugebauerová, J., Raab, S. & Kaffková, K. 2015 Evaluation of content of essential oil in selected Salvia L. species Hodnocení obsahu silice vevybraných druzích rodu Salvia L Acta Fac. Pharm. Univ. Comen. 62 23 30

    • Search Google Scholar
    • Export Citation
  • Palacio, L., Baeza, M.C., Cantero, J.J., Cusidó, R. & Goleniowski, M.E. 2008 In vitro propagation of “Jarilla” (Larrea divaricata CAV.) and secondary metabolite production Biol. Pharm. Bull. 31 2321 2325

    • Search Google Scholar
    • Export Citation
  • Peeters, A.J.M., Gerards, W., Barendse, G.W.M. & Wullems, G.J. 1991 In vitro flower bud formation in tobacco: Interaction of hormones Plant Physiol. 97 402 408

    • Search Google Scholar
    • Export Citation
  • Reddy, N.S., Navanesan, S., Sinniah, S.K., Wahab, N.A. & Sim, K.S. 2012 Phenolic content, antioxidant effect and cytotoxic activity of Leea indica leaves BMC Comp. Alter. Med. 12 128 134

    • Search Google Scholar
    • Export Citation
  • Ryu, S.Y., Lee, C.O. & Choi, S.U. 1996 In vitro cytotoxicity of tanshinones from Salvia miltiorrhiza Planta Med. 63 339 342

  • Shan, C.G., Wang, Z.F., Su, X.H., Yan, S.L. & Sunday, H.C. 2007 Study advance in Salvia miltiorrhiza tissue culture Res. Prac. Chin. Med. 22 54 57 (in Chinese)

    • Search Google Scholar
    • Export Citation
  • Sharma, R.K. & Wakhlu, A.K. 2003 Regeneration of Heracleum candicans wall plants from callus cultures through organogenesis J. Plant Biochem. Biotechnol. 12 71 72

    • Search Google Scholar
    • Export Citation
  • Shi, M., Kwok, K.W. & Wu, J.Y. 2007 Enhancement of tanshinone production in Salvia miltiorrhiza Bunge (red or Chinese sage) hairy-root culture by hyperosmotic stress and yeast elicitor Biotechnol. Appl. Biochem. 46 191 196

    • Search Google Scholar
    • Export Citation
  • Singleton, V.L., Orthofer, R. & Lamuela-Raventós, R.M. 1999 Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent Methods Enzymol. 14 152 178

    • Search Google Scholar
    • Export Citation
  • Surveswaran, S., Cai, Y.Z., Xing, J., Corke, H. & Sunday, M. 2010 Antioxidant properties and principal phenolic phytochemicals of Indian medicinal plants from Asclepiadoideae and Periplocoideae Nat. Prod. Res. 24 206 221

    • Search Google Scholar
    • Export Citation
  • Wang, M., Li, J., Zhang, L., Yang, R.W., Ding, C.B., Zhou, Y.H. & Yin, Z.Q. 2011 Genetic diversity among Salvia miltiorrhiza Bunge and related species using morphological traits and RAPD markers J. Med. Plants Res. 53 197 204

    • Search Google Scholar
    • Export Citation
  • Wang, Q.J., Zheng, L.P., Yuan, H.Y. & Wang, J.W. 2013 Propagation of Salvia miltiorrhiza from hairy root explants via somatic embryogenesis and tanshinone content in obtained plants Ind. Crops Prod. 50 648 653

    • Search Google Scholar
    • Export Citation
  • Wu, J.Y. & Shi, M. 2008 Ultrahigh diterpenoid tanshinone production through repeated osmotic stress and elicitor stimulation in fed batch culture of Salvia miltiorrhiza hairy roots Appl. Microbiol. Biotechnol. 78 441 448

    • Search Google Scholar
    • Export Citation
  • Xanthopoulou, M.N., Fragopoulou, E., Kalathara, K., Nomikos, T., Karantonis, H.C. & Antonopoulou, S. 2010 Antioxidant and anti-inflammatory activity of red and white wine extracts Food Chem. 120 665 672

    • Search Google Scholar
    • Export Citation
  • Xie, X.H., Li, J.H., Feng, W.L., Xie, H.E., Chen, L., Li, H.X. & Wu, C.X. 2004 The rapid multiplication of Salvia miltiorrhiza by tissue culture J. Chin. Med. Mater. 27 474 475 (in Chinese)

    • Search Google Scholar
    • Export Citation
  • Xu, Y. & Xiang, Y.Y. 2014 The measurement of total Danshinone in Salvia miltiorrhiza J Hubei Tradit. Chin. Med. 36 70 71 (in Chinese)

  • Zhang, C., Yain, Q., Cheuk, W.K. & Wu, J.Y. 2004 Enhancement of tanshinone production in Salvia miltiorrhiza hairy root culture by Ag+ elicitation and nutrient feeding Planta Med. 70 147 151

    • Search Google Scholar
    • Export Citation
  • Zhao, J., Chen, Z.S. & Wan, J. 1999 Clonic reproduction and plant regeneration from blade of Salvia miltiorrhiza Bunge J. Cent. China Normal Univ. (Nat. Sci.) 33 108 111 (in Chinese)

    • Search Google Scholar
    • Export Citation
  • Zhao, J.L., Zhou, L.G. & Wu, J.Y. 2010 Promotion of Salvia miltiorrhiza hairy root growth and tanshinone production by polysaccharide–Protein fractions of plant growth-promoting rhizobacterium Bacillus cereus Process Biochem. 45 1517 1522

    • Search Google Scholar
    • Export Citation
  • Zheng, W., Xu, X.D., Dai, H. & Chen, L.Q. 2009 Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species Scientia Hort. 122 138 141

    • Search Google Scholar
    • Export Citation
  • Zhu, Y.Z., Huang, S.H., Tan, B.K.H., Sun, J., Whiteman, M. & Zhu, Y.C. 2004 Antioxidants in Chinese herbal medicines: A biochemical perspective Nat. Prod. Rpt. 21 478 489

    • Search Google Scholar
    • Export Citation

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Contributor Notes

This work was funded by the Natural Science Foundation of Shandong province (ZR2014JL021), the National Natural Science Foundation of China (Grant No. 31700624), and the key construction project for high level of applied science of Shandong province-Bioengineering major (2016) 11-10.

These authors contributed equally to this work.

Corresponding author. E-mail: lblzzkk@126.com.

  • View in gallery

    Micropropagation of Salvia miltiorrhiza. (A) Green-colored protuberances from leaf explants; (B) Adventitious shoots induced after 3 weeks; (C) Shoot proliferation and elongation; (DG) The plantlets rooted from the shoot base; (H) Acclimatized plants in the greenhouse (bars = 1 cm).

  • View in gallery

    Phytochemical contents of extracts from micropropagated and seed-derived Salvia miltiorrhiza plants. Total flavonoid (A) and total phenol (B) contents present in shoot (IS) and root (IR) extracts of micropropagated plants, and shoot (SS) and root (SR) extracts of seed-derived plants. Different capital letters among bars indicate statistically significant differences at P ≤ 0.05, according to Duncan’s multiple range test.

  • View in gallery

    Tanshinone content of extracts from micropropagated and seed-derived Salvia miltiorrhiza plants. The tanshinone content of shoot (IS) and root (IR) extracts of micropropagated plants, and shoot (SS) and root (SR) extracts of seed-derived plants. Different capital letters among bars indicate statistically significant differences at P ≤ 0.05, according to Duncan’s multiple range test.

  • View in gallery

    Free radical scavenging ability of extracts from micropropagated and seed-derived Salvia miltiorrhiza plants. Superoxide (A), hydrogen peroxide (B), 2,2-diphenyl-1-picrylhydrazyl radical (C), and nitric oxide (D) scavenging activities of shoot (IS) and root (IR) extracts of micropropagated plants, and shoot (SS) and root (SR) extracts of seed-derived plants and of ascorbic acid (AA). Different capital letters among bars indicate statistically significant differences at P ≤ 0.05, according to Duncan’s multiple range test.

  • Ahmad, N., Fazal, H., Abbasi, B.H., Rashid, M., Mahmood, T. & Fatima, N. 2010 Efficient regeneration and antioxidant potential in regenerated tissues of Piper nigrum L Plant Cell Tissue Organ Cult. 102 129 134

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  • Caboni, E., Tonelli, M.G., Lauri, P.D., Angeli, S. & Damiano, C. 1999 In vitro shoot regeneration from leaves of wild pear Plant Cell Tissue Organ Cult. 59 1 7

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  • Chen, H., Chen, F., Chiu, F.C.K. & Lo, C.M.Y. 2001 The effect of yeast elicitor on the growth and secondary metabolism of hairy root cultures of Salvia miltiorrhiza Enzyme Microb. Technol. 28 100 105

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  • Claßen-Bockhoff, R., Crone, M. & Baikova, E. 2004 Stamen development in Salvia L.: Homology reinvestigated Intl. J. Plant Sci. 165 475 498

  • Close, D.C. & McArthur, C. 2002 Rethinking the role of many plant phenolic–protection from photodamage not herbivores? Oikos 99 166 172

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  • Frello, S., Venerus, E. & Serek, M. 2002 Regeneration of various species of Crassulaceae, with special reference to Kalanchoë J. Hort. Sci. Biotechnol. 77 204 208

    • Search Google Scholar
    • Export Citation
  • García-Pérez, E., Janet, A., Uribe, G. & García-Lara, S. 2012 Luteolin content and antioxidant activity in micropropagated plants of Poliomintha glabrescens (Gray) Plant Cell Tissue Organ Cult. 108 521 527

    • Search Google Scholar
    • Export Citation
  • Ghimire, B.K., Yu, C.Y. & Chung, I.M. 2012 Direct shoot organogenesis and assessment of genetic stability in regenerants of Solanum aculeatissimum Jacq Plant Cell Tissue Organ Cult. 108 455 464

    • Search Google Scholar
    • Export Citation
  • Gonçalves, S., Fernandes, L. & Romano, A. 2010 High-frequency in vitro propagation of the endangered species Tuberaria major Plant Cell Tissue Organ Cult. 101 359 363

    • Search Google Scholar
    • Export Citation
  • Gould, K.S., Markham, K.R., Smith, R.H. & Goris, J.J. 2000 Functional role of anthocyanins in the leaves of Quintinia serrata A Cunn. J. Expt. Bot. 51 1107 1115

    • Search Google Scholar
    • Export Citation
  • Halliwell, B. & Gutteridge, J.M.C. 2007 Free radicals in biology and medicine, Vol. 3. 4th ed. Oxford Univ. Press, Oxford, UK

  • Hosoki, T. & Tahara, Y. 1993 In vitro propagation of Salvia leucantha Cav HortScience 28 226

  • Hu, Z.B. & Alfermann, A.W. 1993 Diterpenoid production in hairy root cultures of Salvia miltiorrhiza Phytochemistry 32 699 703

  • Ishimary, K., Sudo, H., Satake, M., Matsunaga, Y., Hasegawa, Y., Takemoto, S. & Shimomura, K. 1990 Amarogentin, amaroswerin and four xanthones from hairy root cultures of Swertia japonica Phytochemistry 29 1563 1565

    • Search Google Scholar
    • Export Citation
  • Jagtap, U.B., Waghmare, S.R., Lokhande, V.H., Suprasanna, P. & Bapat, V.A. 2011 Preparation and evaluation of antioxidant capacity of jackfruit (Artocarpus heterophyllus Lam.) wine and its protective role against radiation induced DNA damage Ind. Crops Prod. 34 1595 1601

    • Search Google Scholar
    • Export Citation
  • Kim, D.O., Chun, O.K., Kim, Y.J., Moon, H.Y. & Lee, C.Y. 2003 Quantification of polyphenolics and their antioxidant capacity in fresh plums J. Agr. Food Chem. 51 6509 6515

    • Search Google Scholar
    • Export Citation
  • Kinsella, J.E., Frankel, E., German, B. & Kanner, J. 1993 Possible mechanisms for the protective role of antioxidants in wine and plant foods Food Technol. 47 85 89

    • Search Google Scholar
    • Export Citation
  • Kordi, M., Kaviani, B. & Hashemabadi, D. 2013 In vitro propagation of Kalanchoe blossfeldiana using BA and NAA Eur. J. Expt. Biol. 3 285 288

  • Kumaran, A. & Karunakaran, R.J. 2007 In vitro antioxidant activities of methanol extracts of five Phyllanthus species from India LWT-Food Sci. Technol. 40 344 352

    • Search Google Scholar
    • Export Citation
  • Lan, T.F., Wang, X., Wang, D.J., Wang, L., Liu, Q. & Yu, Z.Y. 2012 Determination of four tanshinones in Salvia miltiorrhiza Radix et Rhizoma with quantitative analysis of multi-components by single-marker Chin. Tradit. Herbal Drugs 43 2420 2423 (in Chinese)

    • Search Google Scholar
    • Export Citation
  • Li, B., Zhang, C.L., Peng, L., Liang, Z.S., Yan, X.J., Zhu, Y.H. & Liu, Y. 2015 Comparison of essential oil composition and phenolic acid content of selected Salvia species measured by GC-MS and HPLC methods Ind. Crops Prod. 69 329 334

    • Search Google Scholar
    • Export Citation
  • Li, Q., Liu, W., Luo, Z.L. & Yang, M.H. 2012 Simultaneous determination of four tanshinones in Salvia miltiorrhiza by QAMS Chian J. Chinese Mater. Med. 37 824 828 (in Chinese)

    • Search Google Scholar
    • Export Citation
  • Liu, B.L., Fang, H.Z., Meng, C.R., Chen, M., Chai, Q.D., Zhang, K. & Liu, S.J. 2017 Establishment of a rapid and efficient micropropagation system for succulent plant Haworthia turgida Haw HortScience 52 1 5

    • Search Google Scholar
    • Export Citation
  • Lystvan, K., Belokurova, V., Sheludko, Y., Ingham, J.L., Prykhodko, V., Kishchenko, O., Paton, E. & Kuchuk, M. 2010 Production of bakuchiol by in vitro systems of Psoralea drupacea Bge Plant Cell Tissue Organ Cult. 101 99 103

    • Search Google Scholar
    • Export Citation
  • Manivannan, A., Soundararajan, P., Halimah, N., Chung, H.K. & Jeong, B.R. 2015 Blue LED light enhances growth, phytochemical contents, and antioxidant enzyme activities of Rehmannia glutinosa cultured in vitro Hort. Environ. Biotechnol. 56 105 113

    • Search Google Scholar
    • Export Citation
  • Menković, N., Fodulović, S.K., Momcilović, I. & Grubisić, D. 2000 Quantitative determination of secoiridoid and gamma-pyrone compounds in Gentiana lutea cultured in vitro Planta Med. 66 96 98

    • Search Google Scholar
    • Export Citation
  • Moncada, S., Palmer, R.M. & Higgs, E.A. 1991 Nitric oxide: Physiology, pathophysiology, and pharmacology Pharmacol. Rev. 43 109 142

  • Neugebauerová, J., Raab, S. & Kaffková, K. 2015 Evaluation of content of essential oil in selected Salvia L. species Hodnocení obsahu silice vevybraných druzích rodu Salvia L Acta Fac. Pharm. Univ. Comen. 62 23 30

    • Search Google Scholar
    • Export Citation
  • Palacio, L., Baeza, M.C., Cantero, J.J., Cusidó, R. & Goleniowski, M.E. 2008 In vitro propagation of “Jarilla” (Larrea divaricata CAV.) and secondary metabolite production Biol. Pharm. Bull. 31 2321 2325

    • Search Google Scholar
    • Export Citation
  • Peeters, A.J.M., Gerards, W., Barendse, G.W.M. & Wullems, G.J. 1991 In vitro flower bud formation in tobacco: Interaction of hormones Plant Physiol. 97 402 408

    • Search Google Scholar
    • Export Citation
  • Reddy, N.S., Navanesan, S., Sinniah, S.K., Wahab, N.A. & Sim, K.S. 2012 Phenolic content, antioxidant effect and cytotoxic activity of Leea indica leaves BMC Comp. Alter. Med. 12 128 134

    • Search Google Scholar
    • Export Citation
  • Ryu, S.Y., Lee, C.O. & Choi, S.U. 1996 In vitro cytotoxicity of tanshinones from Salvia miltiorrhiza Planta Med. 63 339 342

  • Shan, C.G., Wang, Z.F., Su, X.H., Yan, S.L. & Sunday, H.C. 2007 Study advance in Salvia miltiorrhiza tissue culture Res. Prac. Chin. Med. 22 54 57 (in Chinese)

    • Search Google Scholar
    • Export Citation
  • Sharma, R.K. & Wakhlu, A.K. 2003 Regeneration of Heracleum candicans wall plants from callus cultures through organogenesis J. Plant Biochem. Biotechnol. 12 71 72

    • Search Google Scholar
    • Export Citation
  • Shi, M., Kwok, K.W. & Wu, J.Y. 2007 Enhancement of tanshinone production in Salvia miltiorrhiza Bunge (red or Chinese sage) hairy-root culture by hyperosmotic stress and yeast elicitor Biotechnol. Appl. Biochem. 46 191 196

    • Search Google Scholar
    • Export Citation
  • Singleton, V.L., Orthofer, R. & Lamuela-Raventós, R.M. 1999 Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent Methods Enzymol. 14 152 178

    • Search Google Scholar
    • Export Citation
  • Surveswaran, S., Cai, Y.Z., Xing, J., Corke, H. & Sunday, M. 2010 Antioxidant properties and principal phenolic phytochemicals of Indian medicinal plants from Asclepiadoideae and Periplocoideae Nat. Prod. Res. 24 206 221

    • Search Google Scholar
    • Export Citation
  • Wang, M., Li, J., Zhang, L., Yang, R.W., Ding, C.B., Zhou, Y.H. & Yin, Z.Q. 2011 Genetic diversity among Salvia miltiorrhiza Bunge and related species using morphological traits and RAPD markers J. Med. Plants Res. 53 197 204

    • Search Google Scholar
    • Export Citation
  • Wang, Q.J., Zheng, L.P., Yuan, H.Y. & Wang, J.W. 2013 Propagation of Salvia miltiorrhiza from hairy root explants via somatic embryogenesis and tanshinone content in obtained plants Ind. Crops Prod. 50 648 653

    • Search Google Scholar
    • Export Citation
  • Wu, J.Y. & Shi, M. 2008 Ultrahigh diterpenoid tanshinone production through repeated osmotic stress and elicitor stimulation in fed batch culture of Salvia miltiorrhiza hairy roots Appl. Microbiol. Biotechnol. 78 441 448

    • Search Google Scholar
    • Export Citation
  • Xanthopoulou, M.N., Fragopoulou, E., Kalathara, K., Nomikos, T., Karantonis, H.C. & Antonopoulou, S. 2010 Antioxidant and anti-inflammatory activity of red and white wine extracts Food Chem. 120 665 672

    • Search Google Scholar
    • Export Citation
  • Xie, X.H., Li, J.H., Feng, W.L., Xie, H.E., Chen, L., Li, H.X. & Wu, C.X. 2004 The rapid multiplication of Salvia miltiorrhiza by tissue culture J. Chin. Med. Mater. 27 474 475 (in Chinese)

    • Search Google Scholar
    • Export Citation
  • Xu, Y. & Xiang, Y.Y. 2014 The measurement of total Danshinone in Salvia miltiorrhiza J Hubei Tradit. Chin. Med. 36 70 71 (in Chinese)

  • Zhang, C., Yain, Q., Cheuk, W.K. & Wu, J.Y. 2004 Enhancement of tanshinone production in Salvia miltiorrhiza hairy root culture by Ag+ elicitation and nutrient feeding Planta Med. 70 147 151

    • Search Google Scholar
    • Export Citation
  • Zhao, J., Chen, Z.S. & Wan, J. 1999 Clonic reproduction and plant regeneration from blade of Salvia miltiorrhiza Bunge J. Cent. China Normal Univ. (Nat. Sci.) 33 108 111 (in Chinese)

    • Search Google Scholar
    • Export Citation
  • Zhao, J.L., Zhou, L.G. & Wu, J.Y. 2010 Promotion of Salvia miltiorrhiza hairy root growth and tanshinone production by polysaccharide–Protein fractions of plant growth-promoting rhizobacterium Bacillus cereus Process Biochem. 45 1517 1522

    • Search Google Scholar
    • Export Citation
  • Zheng, W., Xu, X.D., Dai, H. & Chen, L.Q. 2009 Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species Scientia Hort. 122 138 141

    • Search Google Scholar
    • Export Citation
  • Zhu, Y.Z., Huang, S.H., Tan, B.K.H., Sun, J., Whiteman, M. & Zhu, Y.C. 2004 Antioxidants in Chinese herbal medicines: A biochemical perspective Nat. Prod. Rpt. 21 478 489

    • Search Google Scholar
    • Export Citation
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